1. Field of the Invention
The present invention generally concerns (1) fluorescent calibration elements generally, but not exclusively, usable with (2) an apparatus, as are commonly but not exclusively used in laboratories, for illuminating and for viewing a macroscopically-sized specimen, for example a mouse, including and most commonly (3) along each of potentially multiple viewing axis. The present invention also generally concerns the methods of locating and using such (1) fluorescent calibration elements.
The present invention particularly concerns the positioning and use of one or more fluorescent calibration elements during any of the illuminating, viewing and or recording of the image of a macroscopically-sized specimen, such as a mouse. The illuminations are potentially along each of multiple viewing axis at a single time, and each such illumination of the specimen along each such axis may be in, potentially, multiple colors (i.e., wavelengths, or frequencies) as serve to excite corresponding fluorescent emissions in the specimen in each of multiple colors (i.e., wavelengths, or frequencies). Moreover, each of the potentially plural induced fluorescent emissions (along each illumination and viewing axis) may be independently controlled in intensity. In particular, multiple fluorescing colored fields as appear within a composite, panoramic, image of the specimen may be—by the adjustability of the fluorescent emissions—both (1) made clearly visible, and (2) balanced one color and area of fluorescent emission to the next—meaning that a bright field of one fluorescent color will not “swamp” a dimmer fluorescent field of another color. Moreover, and nonetheless that the induced fluorescent emissions may be adjusted in intensity—meaning that the dim may be made bright simultaneously that the bright may be made dim—the true and actual intensity of each fluorescent emission may be quantitatively known.
The present invention will be seen to still further concern that all such variable illumination along each of multiple axis as produces multi-color fluorescent emissions of controlled intensity (along each axis, as are individually visible in a composite image) is efficiently realized.
Accordingly, whereas (1) a first related invention regarding panoramic viewing may be simplistically regarded as showing how to comprehensively illuminate and view a macroscopic specimen along a single axis at a single time, and (2) a second invention regarding a fluorescent image calibration step wedge may be simplistically regarded as showing how to quantify each of multiply-colored fluorescent emissions permissively simultaneously appearing in each of multiple (illumination and) viewing axis in a composite, panoramic, image, (3) the present invention shows how efficiently illuminate a macroscopic specimen, permissively along each of multiple axis, with some sophistication to the end to that each of multiple fluorescent emissions induced in the specimen (by the illuminating) will be well and easily viewable. Specifically, the present invention will be seen to regard image illumination for viewing where such illumination is not only realized along each of multiple viewing axis at a single time, but where this axial illumination is readily selectively balanced in either of intensity (and/or, less commonly, color (i.e., wavelength, or frequency)). This selective balancing of illuminations—permissively separately independently along each of a plurality of specimen illumination paths—is so that fluorescence induced in the specimen and appearing in the composite image as different fields having more than one color (i.e., wavelengths, or frequencies) will so appear with roughly equal intensity each color.
Despite this “adjustment” in the intensities of each fluorescent color, the real and true intensity of fluorescence at each color (i.e., wavelength, or frequency), is readily calibrated for each color, and is even so calibratable separately along each illumination path.
2. Description of the Prior Art
2.1 General Laboratory Apparatus and Methods for Illumination and Observation of Macroscopically Sized Specimens
Apparatus to illuminate and to hold macroscopically-sized specimens for viewing, including viewing as may involve the taking of photographs, are known in the art. These apparatus hold secure a macroscopically-sized specimen to be viewed, including for example a live specimen and more particularly a laboratory animal and still more particularly a mouse, upon a specimen stage. A source of illuminating radiation—most commonly a narrowband, colored, light radiation—is brought to bear upon the held specimen.
The illuminating radiation sources may consist of the emitting end of a fiber optic, a fiber optic bundle, or a light pipe or the like. The illuminating radiation itself may, by way of example, be sufficient so as to induce fluorescence in the specimen, including in a specimen as may have been previously fused with fluorescing agents that most commonly serve to make regions of the specimen that are of interest more visible or otherwise detectable.
The illuminated specimen may be, and commonly is, digitally imaged, but may also and/or alternatively be photographed, including in its emitted fluorescent light.
2.2 The Utility of Introducing Quantitative Rigor into Observations of Macroscopically Sized Specimens
The present and related inventions will generally be seen to be directed to a common goal of imparting the imaging, and photographing, of macroscopic specimens (especially specimens as are caused to fluoresce)—a process generally presently conducted “ad hoc”—with a great deal of scientific rigor.
As of present, circa 2004, the images, or photographs produced by conventional illumination and observation of macroscopically-sized specimens, such as the biological specimen of a mouse, tend to be rather crude. Most typically the mouse will be illuminated so that an region of interest, such as a tumor, previously absorbing fluorescent dye will be caused to fluoresce, and the fluorescent region of the resulting image is indicated only that the mouse has the tumor.
In this rudimentary observation many, many things are lacking.
First, it is not possible to view the mouse specimen along multiple axis, or panoramically around a broad angular field, at the same time. This precludes looking at the same tumor in the mouse from two or more different directions, and from looking at multiple tumors as may exist within different regions of the mouse all at the same time.
Accordingly, it would firstly be useful if a single macroscopically-sized specimen, for example a mouse, could be observed along each of multiple axis, for example left side and right side and fore and aft, all at the same time.
Second, no dimensional scale, either linear or grid, typically accompanies the viewed image of the specimen (the mouse). Such a scale is useful for, by way of example, judging the dimension(s) and volume of the observed tumor. Accordingly, it would secondly be useful if the image of a specimen (for example, a mouse) inherently contained a scale of either the linear or the grid type.
Third, the illumination is commonly so as to induced fluorescence of a single fluorescing agent at a single color at a single time. Even though a resulting image of specimen, which is normally preserved as a photograph, may be in color, for example of a green fluorescing region within a white mouth, the image, and photograph, really contains no more information than a black and white photograph. This simplistic observation obviates the possibility that a single specimen should contain multiple fluorescing agents which fluoresce at different colors so as to identify corresponding regions of interest within the (single) specimen. This simplistic observation does not make optimal use of modern color digital cameras.
Although multi-color photographs of multiple fluorescent colors within a single specimen may in the past have been made, any such images would likely have been derived by illumination with a single light sufficient so as to induce emission in each of multiple fluorescent agents. Otherwise the “plumbing” of excitation lights to the specimen may become unwieldy. To the best knowledge of the inventor, it has not been commonly thought to simultaneously illuminate a macroscopic specimen with multiple colors (as are targeted to induce associated multiple fluorescent emissions), let alone to attempt adjustment of the intensity of each color within a resulting composite image.
In other words, a body impregnated with fluorescent red dye may appear to fluoresce red light quite brightly while another body (or the same body or portion thereof as may have picked up green fluorescent dye at a different time and/or to a different extent) may, under the same common illumination, fluoresce green light quite dimly. Nonetheless that the body, or tumor, fluorescing red shows brightly in the image, and the body, or tumor, fluorescing green shows but dimly in the image, the “green” tumor or stage may be of equal size and/or interest to the “red” tumor. What looks bright, and what looks dim, in the composite image is, of course, a function of the efficiency of the uptake of the fluorescent dyes, the efficiency of the illumination excitation of each, and the efficiency of each dye to fluoresce, among other factors. Thus, even should multiple illumination sources of different frequencies be simultaneously “optically plumbed” to illuminate the macroscopic body under observation, independently adjusting selecting illumination frequencies and adjusting the intensity of each so that the resulting “red” tumor and “green” tumor images in the composite are somewhat comparable.
Accordingly, it would thirdly be useful if each of multiple regions fluorescing at different colors within a single composite image of a specimen (for example, a mouse) could be independently adjusted in intensity, clearly rendering visible in the composite image those things and/or regions that the researcher and image taker desires to be well seen, while suppressing within the composite image other things and/or regions that are deemed unimportant. It would be especially useful if this selective differential “highlighting” of each of multiple colors of fluorescent emission could somehow be realized from but a single, common, illuminating light source.
Some little thought will reveal, however, that should such control be given to the image maker, then it may soon become impossible to know what has been done in manipulation of the composite image and its colors, and to know what imaged things and/or regions “really” look like under normal conditions. It thus, fourth, problematic that no scale of the intensity(ies) of (potentially several different) fluorescent emission(s) typically accompanies the viewed image of the specimen (the mouse). Such a scale is useful for, by way of example, judging how bright or how dim were things and/or regions—nonetheless to their appearance within the composite image—under normal, and standard, illumination conditions.
Accordingly, it would fourthly be useful if the image of a specimen (for example, a mouse) inherently contained a scale of by which any of the intensity(ies), color(s), or, as even more exotic criteria seldom useful, radiation temperature. The color scale might be broken down into hue, chroma (purity, or saturation) and brightness (value). In this manner a viewer of a composite image might be able to say: “I see by comparison to a scale that is within the selfsame image that this clearly visible first object (or area) fluoresced red, and that it was in fact quite bright, even to the point of obscuration, until intentionally diminished in intensity. Meanwhile I also see by comparison to another portion of the same scale, or another scale also contained within the image, that this equally clearly visible second object (or area) fluoresced green, but only dimly so, and that this second object has intentionally been accentuated in intensity by action of the image maker.”
2.3 Definitions of “Optical Density” and “Optical Transmittance”
The fluorescent calibration step wedge of the present invention will be seen to vary in optical density (OD) and optical transmittance (T).
By definition, optical density (OD) is, for a given wavelength, an expression of the transmittance of an optical element. Optical density is expressed by log 10(1/T) where T is transmittance. The higher the optical density, the lower the transmittance. Optical density times 10 is equal to transmission loss expressed in decibels, e.g., an optical density of 0.3 corresponds to a transmission loss of 3 dB.
Also by definition, transmittance is the ratio of the transmitted power to the incident power. In optics, transmittance is usually expressed as optical density or in percent. Transmittance was formerly called “transmission.”
2.4 Quantum Dots
The fluorescent calibration step wedge of the present invention will also be seen to optionally employ quantum dots.
The following abbreviated explanation of quantum dots is in accordance with the paper “Probing the Optical Properties of Single Quantum Dots” by Jeffrey R. Krogmeier, Jeeseong Hwang, & Lori S. Goldner, National Institute of Standards and Technology, Optical Technology Division, Physics Laboratory, 100 Bureau Drive, Mail Stop 8441, Gaithersburg, Md. 20899 USA
Semiconductor nanocrystals or quantum dots are gaining interest as fluorescent tags for biological molecules due to their large quantum yield and photostability. Quantum dots are semiconductor crystallites 2 nm to 10 nm in diameter that contain approximately 500–1000 atoms of materials as cadmium and selenide. Quantum dots fluoresce with a broad absorption spectrum and a narrow emission spectrum. The larger the quantum dot the longer wavelength emitted. The broad absorption spectrum allows many different quantum dots to be excited with one excitation source. The emission spectra for each dot is typically very narrow, on the order of 30 nanometers, which permits spectral resolution of adjacent dots.
Quantum dots are sometimes employed as biological tags. In order to employ quantum dots as biological tags, the nanocrystal must be water soluble and capable of being conjugated to the biological molecule of interest. To accomplish this, much effort has been dedicated to functionalizing the nanocrystal surface with water-soluble, reactive chemical moieties. To employ quantum dots in biological assays, the optical properties of functionalized quantum dots must be understood. In the approach of the subject paper, single molecule confocal microscopy is used to probe the fluorescent properties of functionalized quantum dots at the single particle level. Others have shown that unfunctionalized or bare quantum dots demonstrate fluorescence intermittency or blinking on the millisecond timescale. Carboxylated, amine activated, and bare quantum dots are all useful in understanding the effect of quantum dot coatings on the optical properties.